Biological membranes play a multi-faceted role in cell biology. In addition to providing the physical barrier that defines a cell or organelle, biological membranes play a role in the cellular processes including the transport of nutrients and waste products, the maintenance of homeostasis within a cell or organelle, intercellular and intracellular signaling, environmental sensing, cellular reproduction, and cellular motility. Assays for components or activities associated with a biological membrane are used both to identify and measure particular types of cells (e.g., in cell typing, cell counting, and assays for pathogens such as bacteria and virus particles) and in basic biological studies of membrane function. The importance of membrane-associated proteins, lipids and sugars in cellular signaling has also made them important targets for the screening of drugs that affect signaling pathways.
Drug screening assays are often carried out using highly purified membrane-free preparations. For example, screens for inhibitors of a membrane receptor-ligand binding interaction often involve the use of purified detergent-solubilized membrane receptor proteins (or, alternatively, truncated membrane receptor proteins missing their trans-membrane domains). There is a trend, however, towards conducting assays using membrane components in their native state (i.e., assays employing tissue, whole cells or organelles, natural membrane fragments or vesicles, or membrane components reconstituted into synthetic membranes). Assays conducted using these types of complex reagents have several advantages including: i) membrane components are more likely to have their in vivo characteristics when associated with a membrane; ii) the use of membrane-bound reagents facilitates the monitoring of interactions involving more than one membrane-associated component; and iii) the use of membrane-bound reagents facilitates the study of membrane-bound activities (e.g., the measurement of down stream signaling events controlled by a membrane-associated receptor).
At this time, there are a number of commercially available instruments that utilize electrochemiluminescence (ECL) for analytical measurements including drug screening. Species that can be induced to emit ECL (ECL-active species) have been used as ECL labels. Examples of ECL labels include: i) organometallic compounds where the metal is from, for example, the noble metals of group VIII, including Ru-containing and Os-containing organometallic compounds such as the tris-bipyridyl-ruthenium (RuBpy) moiety and ii) luminol and related compounds. Species that participate with the ECL label in the ECL process are referred to herein as ECL coreactants. Commonly used coreactants include tertiary amines (e.g., see U.S. Pat. No. 5,846,485, herein incorporated by reference), oxalate, and persulfate for ECL from RuBpy and hydrogen peroxide for ECL from luminol (see, e.g., U.S. Pat. No. 5,240,863, herein incorporated by reference). The light generated by ECL labels can be used as a reporter signal in diagnostic procedures (Bard et al., U.S. Pat. No. 5,238,808, herein incorporated by reference). For instance, an ECL label can be covalently coupled to a binding agent such as an antibody, nucleic acid probe, receptor or ligand; the participation of the binding reagent in a binding interaction can be monitored by measuring ECL emitted from the ECL label. Alternatively, the ECL signal from an ECL-active compound may be indicative of the chemical environment (see, e.g., U.S. Pat. No. 5,641,623 which describes ECL assays that monitor the formation or destruction of ECL coreactants, herein incorporated by reference). For more background on ECL, ECL labels, ECL assays and instrumentation for conducting ECL assays see U.S. Pat. Nos. 5,093,268; 5,147,806; 5,324,457; 5,591,581; 5,597,910; 5,641,623; 5,643,713; 5,679,519; 5,705,402; 5,846,485; 5,866,434; 5,786,141; 5,731,147; 6,066,448; 6,136,268; 5,776,672; 5,308,754; 5,240,863; 6,207,369 and 5,589,136 and Published PCT Nos. WO99/63347; WO00/03233; WO99/58962; WO99/32662; WO99/14599; WO98/12539; WO97/36931 and WO98/57154, each of which are herein incorporated by reference.
Commercially available ECL instruments have demonstrated exceptional performance. They have become widely used for reasons including their excellent sensitivity, dynamic range, precision, and tolerance of complex sample matrices. The commercially available instrumentation uses flow cell-based designs with permanent reusable flow cells. Recently, ECL instrumentation has been disclosed that uses reagents immobilized on the electrode used to induce ECL (see, e.g., U.S. Pat. No. 6,207,369 and Published PCT Application No. WO98/12539). Multi-well plates having integrated electrodes suitable for such ECL measurements have also been recently disclosed (see, e.g., copending Provisional Application No. 60/301,932 (entitled “Assay Plates, Reader Systems and Methods for Luminescence Test Measurements”, filed on Jun. 29, 2001, hereby incorporated by reference).
The use of multi-well assay plates allows for the parallel processing and analysis of multiple samples distributed in multiple wells of a plate. Typically, samples and reagents are stored, processed and/or analyzed in multi-well assay plates (also known as microplates or microtiter plates). Multi-well assay plates can take a variety of forms, sizes and shapes. For convenience, some standards have appeared for some instrumentation used to process samples for high throughput assays. Assays carried out in standardized plate formats can take advantage of readily available equipment for storing and moving these plates as well as readily available equipment for rapidly dispensing liquids in and out of the plates. Some well established multi-well plate formats include those found on 96-well plates (12×8 array of wells), 384-well plates (24×16 array of wells) and 1536-well plate (48×32 array of well). The Society for Biomolecular Screening has published recommended microplate specifications for a variety of plate formats, the recommended specifications hereby incorporated by reference.